1. Field of the Invention
The invention generally concerns the field of molecular biology. More specifically, the invention relates to methods and compositions for efficient expression of polypeptides comprising disulfide linkages.
2. Description of Related Art
Oxidative protein folding involves two complementary but competing processes: cysteine thiol oxidation, and isomerization of non-native disulfide bonds. The limiting step in the folding of multi disulfide eukaryotic proteins in the bacterial periplasm is often the isomerization of non-native disulfides.
The bacterial disulfide bond formation (“Dsb”) protein family consists of two distinct pathways, DsbA-DsbB, and DsbC/DsbG-DsbD, involved in the formation of disulfides and in the rearrangement of incorrectly formed bonds, respectively (Kadokura et al., 2003; Collet and Bardwell, 2002). The extreme oxidizing nature of mature DsbA (SEQ ID NO:3) mediates rapid oxidation of substrate cysteines, which results in the formation of non-native disulfides, in turn rearranged by DsbC. Consequently, despite the strong oxidizing environment of the periplasmic space, DsbC has to be maintained in a reduced state to interact with the substrate oxidized cysteines (Kadokura et al., 2003; Collet and Bardwell, 2002). To carry on their catalytic activities, DsbA and DsbC are maintained, respectively, in entirely oxidized and reduced states (Ramm and Pluckthun, 2001). DsbA is recycled by the membrane protein DsbB, whereas DsbC is maintained in the reduced state by the membrane protein, DsbD (Arie et al., 2001). Interactions between the two pathways are strongly prevented by kinetic constraints (Rozhkova et al., 2004). As a result, the strong thiol oxidant DsbA, and the strong thiol reductant DsbC, despite coexisting in the same cellular environment, do not appear to exchange electrons with each other, but instead act synergistically during oxidative protein folding. Remarkably, the catalytic domains of DsbC and DsbA show a considerable degree of structural homology, and they both contain a CXXC thioredoxin active site motif for the catalysis of disulfide exchange reactions.
The folding of at least three native proteins, namely the periplasmic acid phosphatase AppA or phytase, the peptidoglycan amidase MepA, and RNase I have been shown to depend on the presence of DsbC (Berkmen et al., 2005). In addition, the folding of a number of heterologous proteins has been shown to require overexpression of DsbC (Kadokura et al., 2003; Collet and Bardwell, 2002; Kim et al., 2004; Kurokawa et al., 2001). It was previously shown that the yield of active vtPA, a truncated version of human tissue plasminogen activator containing 9 disulfide bonds, depends on the DsbC expression level (Qiu et al., 1998).
Zhao et al. (2003) created several engineered polypeptides containing an N-terminal DsbC domain joined to a C-terminal domain from thioredoxin (Trx), DsbA, or portions of protein-disulfide isomerase (PDI). These polypeptides displayed limited isomerase and reductase activities while retaining DsbC-related sequences. It was suggested that the basis of the catalytic activity of DsbC resides in its V-shaped dimeric structure, which allows for the formation of a hydrophobic substrate binding cleft with chaperone activity, and in the presence of two catalytic thioredoxin domains (Segatori et al., 2004). The hybrid DsbC-DsbA or DsbC-TrxA polypeptides described in Segatori et al., 2004, in which the catalytic domain of DsbC had been replaced with DsbA, also displayed disulfide bond formation and isomerase activity, and afforded vtPA yields comparable to those obtained when overexpressing wild-type DsbC under the same conditions. However, the chimeric proteins of Zhao et al. and Segatori et al. each retain DsbC-derived sequences.
The E. coli periplasm contains two classes of enzymes that assist the folding of proteins by catalyzing covalent modification: enzymes that catalyze the reduction and oxidation of disulfide bonds (the Dsb family), and enzymes that catalyze cis/trans peptidyl-prolyl isomerization reactions (PPIases) (Baneyx and Mujacic, 2004). Among the PPIases (E.C. 5.2.1.8), which include SurA (Behrens et al., 2001) and the FK506 binding proteins (FKBP's), an FkpA (e.g., GenBank L28082) has been recently biochemically characterized, and its crystal structure has been solved (Saul et al., 2004). FkpA is a homodimeric V-shaped protein (e.g., FIG. 1) which exhibits a similar gross topology to DsbC. Each monomer in FkpA is formed by an N-terminal dimerization domain and a C-terminal catalytic domain, joined by a long α-helical linker. The two domains are each structurally and functionally independent. The dimerization domains form a binding pocket for the interaction with the substrate, and have been shown to have chaperone activity (Saul et al., 2004; Ramm and Pluckthun, 2001; Arie et al., 2001). FkpA and DsbC lack substantial amino acid sequence identity (e.g., using BLAST with default parameters). There exists a need for methods to catalyze disulfide bond formation and isomerization in bacteria without the expression of a DsbC-containing protein.